Long-Term Depression-Inducing Low Frequency Stimulation Enhances p-Tau181 and p-Tau217 in an Age-Dependent Manner in Live Rats

Abstract

Background: Cognitive decline in Alzheimer's disease (AD) correlates with the extent of tau pathology, in particular tau hyperphosphorylation, which is strongly age-associated. Although elevation of cerebrospinal fluid or blood levels of phosphorylated tau (p-Tau) at residues Thr181 (p-Tau181), Thr217 (p-Tau217), and Thr231 (p-Tau231) are proposed to be particularly sensitive markers of preclinical AD, the generation of p-Tau during brain activity is poorly understood.

Objective: To study whether the expression levels of p-Tau181, p-Tau217, and p-Tau231 can be enhanced by physiological synaptic long-term depression (LTD) which has been linked to the enhancement of p-Tau in hippocampus.

Methods: In vivo electrophysiology was performed in urethane anesthetized young adult and aged male rats. Low frequency electrical stimulation (LFS) was used to induce LTD at CA3 to CA1 synapses. The expression level of p-Tau and total tau was measured in dorsal hippocampus using immunofluorescent staining and/or western blotting.

Results: We found that LFS enhanced p-Tau181 and p-Tau217 in an age-dependent manner in the hippocampus of live rats. In contrast, phosphorylation at residues Thr231, Ser202/Thr205, and Ser396 appeared less sensitive to LFS. Pharmacological antagonism of either N-methyl-D-aspartate or metabotropic glutamate 5 receptors inhibited the elevation of both p-Tau181 and p-Tau217. Targeting the integrated stress response, which increases with aging, using a small molecule inhibitor ISRIB, prevented the enhancement of p-Tau by LFS in aged rats.

Conclusion: Together, our data provide a novel in vivo means to uncover brain plasticity-related cellular and molecular processes of tau phosphorylation at key sites in health and aging.

Keywords: Aging; Alzheimer’s disease; integrated stress response; long-term depression; tau phosphorylation.

Fig. 1

LFS promotes p-Tau181 in an age-dependent manner in live rats. A) Schematic of the field EPSP recording configuration in CA1 stratum radiatum (REC) overlaid with a schematic of a bipolar stimulation electrode (STIM) for Schaffer collateral axon fibers (black) in the ipsilateral hemisphere. Additional excitatory projections from CA3 include local recurrent connections (blue) of CA3 pyramidal cells onto other CA3 pyramidal cells, and the back projection (pink) of CA3 pyramidal neurons to the dentate gyrus (DG). B) Application of LFS (horizontal bar, LFS-900; 900 pulses at 1Hz) induced robust LTD at CA3-CA1 synapses in anaesthetized rats at two different ages (2-3-months and 17-18-months). Calibration bars: vertical, 2 mV; horizontal, 10 ms. C) Summarized EPSP amplitude 30 min post LFS. The EPSP decreased to 63.8±7.8% in 2-3-month-old rats (n = 5, p = 0.0147 compared with Pre), and 62.4±3.4% in 17-18-month-old rats (n = 7, p < 0.0001 compared with Pre) respectively; paired t test. The amplitude of LTD is comparable in both groups (two-way ANOVA, age, F (1, 10) = 0.09427, p = 0.7651). D) The upper panel shows p-Tau181 (red) immunofluorescent staining in dorsal hippocampus (Scale bar: 200μm), CA1, CA3, and hilus of DG (scale bars: 50μm) from 2-3-month-old rats. The corresponding statistical results compared with contralateral side are displayed in the lower panel. The expression level of p-Tau181 was not affected by LFS in dorsal hippocampus (p = 0.4611), CA1 (p = 0.3792), CA3 (p = 0.6842), and DG (p = 0.1953); paired t test. E) Immunofluorescent staining of p-Tau181 (red) in ipsilateral dorsal hippocampus, CA1, CA3, and DG from 17-18-month-old rats. LTD induction by LFS significantly enhanced the level of p-Tau181 in dorsal hippocampus (p < 0.0001), CA1 (p = 0.0220), CA3 (p = 0.0059), and DG (p = 0.0202); paired t test. Values are mean±s.e.m.

Fig. 2

LFS promotes p-Tau217 in an age-dependent manner in live rats. A) The upper panel shows immunofluorescent staining of p-Tau217 (red) in dorsal hippocampus (Scale bar: 200μm), CA1, CA3, and hilus of DG (scale bars: 50μm) from 2-3-month-old rats. The corresponding statistical results compared with contralateral side are displayed in the lower panel. LFS did not affect the expression level of p-Tau217 in dorsal hippocampus (p = 0.3820), CA1 (p = 0.4488), CA3 (p = 0.3409), and DG (p = 0.1567); paired t test. B) Immunofluorescent staining of p-Tau217 (red) in dorsal hippocampus, CA1, CA3, and DG from 17-18-month-old rats. LFS ipsilaterally enhanced the level of p-Tau217 in dorsal hippocampus (p < 0.0019), CA1 (p = 0.0153), CA3 (p = 0.0003), and DG (p = 0.0251); paired t test. Values are mean±s.e.m.

Fig. 3

LFS enhances p-Tau181 and p-Tau217 in aged rats. A) Application of LFS-900 induced robust LTD at CA3-CA1 synapses in anaesthetized 17-18-month-old rats. Calibration bars: vertical, 2 mV; horizontal, 10 ms. B) Summarized EPSP amplitude 30 min post LFS. The EPSP decreased to 54.8±10.2% (n = 5, p = 0.0110 compared with Pre, paired t). C) Left panels show representative blotting band of p-Tau181 and total tau (Tau5) in the hippocampus of age-matched naïve control group and the experimental group either contralateral or ipsilateral to LFS. Statistical results of p-Tau181 over total tau was quantified and normalized to naïve control (n = 5 per group, ipsilateral versus naïve, p = 0.0292; contralateral versus naïve, p = 0.5075; contralateral versus ipsilateral, p = 0.4022; one-way ANOVA-Bonferroni). D) Left panels show representative blotting band of p-Tau217 and total tau (tau5) in age-matched naïve control group, contralateral hippocampus, and ipsilaterally stimulated hippocampus. Statistical results of p-Tau217 over total tau was quantified and normalized to naïve control (n = 5 per group, ipsilateral versus naïve, p = 0.0049; contralateral versus naïve, p = 0.0979; contralateral versus ipsilateral, p = 0.3905; one-way ANOVA-Bonferroni). Values are mean±s.e.m.

Fig. 4

Antagonists of either NMDAR or mGluR5 prevent the elevation of both p-Tau181 and p-Tau217 induced by LFS in aged rats. A) Systemic injection of the competitive NMDAR antagonist CPP (10 mg/kg, i.p.) alone 1 h prior to the application of LFS did not affect LTD induction in 17-18-month-old rats. Similarly, intraperitoneal injection of the mGluR5 negative allosteric modulator MTEP (3 mg/kg) alone did not affect LTD induction by LFS 1 h later in 17-18-month-old rats. Calibration bars: vertical, 2 mV; horizontal, 10 ms. B) Summary of the mean EPSP amplitude pre and post -LFS. The EPSP decreased to 70.2±2.5% (n = 6, p < 0.0001 compared with pre, paired t test) and 79.6±3.7% (n = 5, p = 0.0068 compared with pre, paired t test) 30 min post-LFS in CPP or MTEP treated rats respectively (two-way RM ANOVA, Treatment F_1,9 = 6.999, p = 0.0267). C) The upper panel shows the fluorescent images of p-Tau181 labeling (red) and the lower panel displays the corresponding statistical results in CPP-treated 17-18-month-old rats (Scale bar = 200μm in dorsal hippocampus; scale bars = 50μm in CA1, CA3, and DG regions). LTD induction by LFS did not affect the level of p-Tau181 in dorsal hippocampus (p = 0.1218), CA3 (p = 0.6147), and DG (p = 0.0578) except for CA1 region (p = 0.0353); paired t test. D) The upper panel shows the fluorescent images of p-Tau217 labeling (red) and the corresponding statistical results are displayed in the lower panel in CPP-treated 17-18-month-old rats. No significant difference was found in p-Tau217 levels compared with that in contralateral dorsal hippocampus (p = 0.8075), CA1 (p = 0.0920), CA3 (p = 0.5811), and DG (p = 0.7041); paired t test. E) In MTEP-treated aged rats, LFS failed to promote the levels of p-Tau181 (red as shown in the upper panel). Summarized mean fluorescence intensities (fold to contralateral) in dorsal hippocampus (p = 0.9154), CA1 (p = 0.3161), CA3 (p = 0.1839), and DG (p = 0.8417); paired t test. F) MTEP treatment also prevented the enhancement of p-Tau217 by LFS in aged rats. The upper panel shows the fluorescent images of p-Tau217 (red). The corresponding statistical results are displayed in the lower panel and no significant difference was found in dorsal hippocampus (p = 0.7702), CA1 (p = 0.1598), CA3 (p = 0.2262), and DG (p = 0.5311); paired t test. Values are mean±s.e.m.

Fig. 5

ISRIB blocks LFS-induced enhancement of p-Tau181 and p-Tau217 in aged rats. A) Experimental paradigm for ISRIB injections and electrophysiology experiments. ISRIB (2.5 mg/kg, i.p.) was systemically injected for 3 days and in vivo electrophysiology experiments were performed 18 days after the last injection. B) LFS induced robust LTD in ISRIB-treated aged rats. Calibration bars: vertical, 2 mV; horizontal, 10 ms. C) The EPSP decreased to 81.8±2.7% at 30 min post LFS (n = 4, p = 0.0211 compared with pre, paired t test). D) Treatment of ISRIB completely prevented the increase of p-Tau181 induced by LFS in aged rats. The upper panel shows the fluorescent images of p-Tau181 labeling (red). As summarized in the lower panel, no significant difference was found in the dorsal hippocampus (p = 0.0579), CA1 (p = 0.7006), CA3 (p = 0.0715), and DG (p = 0.2696); paired t test. E) Treatment with ISRIB also successfully blocked the enhancement of p-Tau217 induced by LFS in aged rats. The upper panel shows the fluorescent images of p-Tau217 labeling (red). Summarized statistical results in the lower panel show no significant difference in all regions, including dorsal hippocampus (p = 0.1185), CA1 (p = 0.9049), CA3 (p = 0.9172), and DG (p = 0.6636); paired t test. Scale bar = 200μm in hippocampus, scale bar = 50μm in CA1, CA3 and DG regions. F) Schematic diagram showing proposed mechanism of LTD-inducing LFS-triggered tau phosphorylation in aged rats. The ISR is enhanced in aged brain, resulting in increased susceptibility to cellular processes underlying the induction of mGluR and NMDAR-dependent LTD either directly or indirectly. Facilitation of certain LTD-associated kinases, such as GSK3α/β or Cdk5, may phosphorylate tau at residues Thr181 and Thr217 with high sensitivity. Values are mean±s.e.m.